Evaluation of Viral Inactivation and Survival in Three Unique Environments, through the Use of MS2 Coliphage as a Surrogate

Abstract

Surrogate organisms have been used to study highly pathogenic organisms, or organisms that cannot be cultured in the laboratory. Surrogates are selected based on multiple similarities to the pathogen, such as morphology, genome size and structure, and environmental characteristics. This dissertation utilized MS2 coliphage as a surrogate for norovirus and Ebola virus in three environments. MS2 is an icosahedral, single-stranded RNA bacteriophage. It is a male-specific coliphage that infects the bacteria Escherichia coli. Its properties, such as morphology and survival in the environment, have been likened to those of many enteric viruses. Because of this, it has been used as a surrogate for pathogenic enteric viruses for disinfection testing on surfaces, in water and in food; modeling the movement and survival of pathogens in different environments; and transfer properties from surfaces. This dissertation utilized MS2 as a surrogate in three different studies. In the first, MS2 is used as a surrogate for human enteric viruses in irrigation canals to predict the re-suspension of pathogenic viruses from bed sediment into overlying irrigation water using a flume to re-create field conditions in the laboratory. MS2 re-suspension into the overlying water was characterized at varying flow rates and velocities using two sediment types. Its overall re-suspension was not statistically significantly different (p > 0.05) between flow rates. The additional studies in this dissertation used MS2 as a surrogate for Ebola virus in human waste. Ebola virus is a BSL-4 organism that is spread through direct contact with bodily fluids. It is found in bodily fluids in concentrations between 10^5.5 and 10⁸ genome copies per milliliter. In the first study using MS2 as a surrogate for Ebola virus, efficacies of four disinfectants were tested using 10¹² PFU of MS2 in one liter containing 2.25% (w/v) organic matter at three contact times (1, 15 and 30 minutes). The purpose of this study was to assess the disinfectants on reducing virus in waste before toilet flushing. Peracetic acid and quaternary ammonium formulation were found to reduce the concentration of MS2 in the toilet bowl the fastest (within one minute) with the greatest reduction (2.26 and 1.99 log₁₀), when compared with the other disinfectants. Reductions observed from hydrogen peroxide were significantly less than those from peracetic acid and quaternary ammonium (p < 0.05). The contamination of restroom surfaces by MS2 was also evaluated after toilet flushing with and without disinfectant treatment. All four disinfectants were found to significantly reduce the viral concentrations on fomites after 15 minutes of contact (p < 0.05). Despite disinfectant use, three sites were contaminated in 100% of trials (N = 18). These were the toilet bowl rim, the toilet seat top and underside. The final study evaluated the inactivation of MS2 and several other viruses by thermophilic and mesophilic anaerobic digestion. Little information is available on the influence of the wastewater treatment process, specifically anaerobic digestion, on emerging viruses, such as Ebola virus. It is important to evaluate this process due to the environmental disposal and discharge of wastewater and solids into the environment. All viruses were recoverable after mesophilic digestion (reductions from 1.8-6.6 log₁₀ per mL), except the lipid-containing bacteriophage Φ6. Thermophilic digestion inactivated all viruses significantly (p = 0.0011) more than mesophilic digestion. The reductions by thermophilic digestion ranged from 2.8-7.1 log₁₀ per mL. The inactivation between the initial concentration and both digestion types was statistically significant (p = 0.007).